In vitro and in vivo formation of aminophenylnorharman from norharman and aniline

Abstract Norharman is not mutagenic to Salmonella strains, but becomes so to S. typhimurium TA98 and YG1024 with S9 mix in the presence of the non-mutagenic aromatic amine, aniline. The mutagenicity from norharman and aniline in the presence of S9 mix is reported to be due to formation of a mutagenic compound, 9-(4′-aminophenyl)-9 H -pyrido[3,4- b ]indole (aminophenylnorharman, APNH). In the present study, we examined which enzymes might be involved in in vitro formation of APNH from norharman and aniline. When norharman (5 mg) and aniline (2.5 mg) were incubated with S9 mix, the S9 fraction of which was prepared from the livers of rats treated with phenobarbital (PB) and β-naphthoflavone (β-NF), at 37 °C for 20 min, 496 ng of APNH was produced. Formation of APNH was also detected with the microsomal fraction, but not with the cytosol fraction. The addition of a P450 inhibitor, SKF-525A, to the reaction mixture at a dose of 5 mM resulted in a decrease of APNH formation, to around 40% of the control level. These results suggest involvement of P450 enzyme(s) in the formation of APNH from norharman and aniline. Moreover, a microsomal fraction from human liver was demonstrated to have the capacity to produce APNH from norharman and aniline, similar to the case with the microsomal fraction from rat liver. When norharman (90 mg/kg) and aniline (90 mg/kg) were administered to rats by gavage, APNH could be detected in the urine, at a rate of 19.6 ng ±16.9  ng per 24 h. The level was increased by treatment of the urine samples with hydrochloric acid, suggesting some APNH was excreted into urine as conjugated forms. Thus, it is likely that APNH may be produced from norharman and aniline in the human body.